Abscisic acid (ABA) plays an important role in fruit development and ripening. Here, three NCED genes encoding 9-cis-epoxycarotenoid dioxygenase (NCED, a key enzyme in the ABA biosynthetic pathway) and three CYP707A genes encoding ABA 8′-hydroxylase (a key enzyme in the oxidative catabolism of ABA) were identified in tomato fruit by tobacco rattle virus-induced gene silencing (VIGS). Quantitative real-time PCR showed that VIGS-treated tomato fruits had significant reductions in target gene transcripts. In SlNCED1-RNAi-treated fruits, ripening slowed down, and the entire fruit turned to orange instead of red as in the control. In comparison, the downregulation of SlCYP707A2 expression in SlCYP707A2-silenced fruit could promote ripening; for example, colouring was quicker than in the control. Silencing SlNCED2/3 or SlCYP707A1/3 made no significant difference to fruit ripening comparing RNAi-treated fruits with control fruits. ABA accumulation and SlNCED1transcript levels in the SlNCED1-RNAi-treated fruit were downregulated to 21% and 19% of those in control fruit, respectively, but upregulated in SlCYP707A2-RNAi-treated fruit. Silencing SlNCED1 or SlCYP707A2 by VIGS significantly altered the transcripts of a set of both ABA-responsive and ripening-related genes, including ABA-signalling genes (PYL1, PP2C1, and SnRK2.2), lycopene-synthesis genes (SlBcyc, SlPSY1 and SlPDS), and cell wall-degrading genes (SlPG1, SlEXP, and SlXET) during ripening. These data indicate that SlNCED1 and SlCYP707A2 are key genes in the regulation of ABA synthesis and catabolism, and are involved in fruit ripening as positive and negative regulators, respectively.

Rch Pa
KaiJi
1
WenbinKai
1
BoZhao
1
YufeiSun
1
BingYuan
0
ShengjieDai
1
QianLi
1
PeiChen
1
YaWang
1
YuelinPei
1
HongqingWang
1
YangdongGuo
1
PingLeng
1
0
Department of Chemistry and Biochemistry, University of Arizona, 1306 East University BouleVard
, Tucson,
USA
1
College of Agronomy and Biotechnology, China Agricultural University
,
Beijing 100193, PR China
Abscisic acid (ABA) plays an important role in fruit development and ripening. Here, three NCED genes encoding 9-cis-epoxycarotenoid dioxygenase (NCED, a key enzyme in the ABA biosynthetic pathway) and three CYP707A genes encoding ABA 8-hydroxylase (a key enzyme in the oxidative catabolism of ABA) were identified in tomato fruit by tobacco rattle virus-induced gene silencing (VIGS). Quantitative real-time PCR showed that VIGS-treated tomato fruits had significant reductions in target gene transcripts. In SlNCED1-RNAi-treated fruits, ripening slowed down, and the entire fruit turned to orange instead of red as in the control. In comparison, the downregulation of SlCYP707A2 expression in SlCYP707A2-silenced fruit could promote ripening; for example, colouring was quicker than in the control. Silencing SlNCED2/3 or SlCYP707A1/3 made no significant difference to fruit ripening comparing RNAi-treated fruits with control fruits. ABA accumulation and SlNCED1transcript levels in the SlNCED1-RNAi-treated fruit were downregulated to 21% and 19% of those in control fruit, respectively, but upregulated in SlCYP707A2-RNAi-treated fruit. Silencing SlNCED1 or SlCYP707A2 by VIGS significantly altered the transcripts of a set of both ABA-responsive and ripening-related genes, including ABA-signalling genes (PYL1, PP2C1, and SnRK2.2), lycopene-synthesis genes (SlBcyc, SlPSY1 and SlPDS), and cell wall-degrading genes (SlPG1, SlEXP, and SlXET) during ripening. These data indicate that SlNCED1 and SlCYP707A2 are key genes in the regulation of ABA synthesis and catabolism, and are involved in fruit ripening as positive and negative regulators, respectively.
-
Abscisic acid (ABA) plays an important role in plant growth,
stomatal movement, seed dormancy, and germination
(Melcher et al., 2009; Nishimura et al., 2009). Moreover, it
mediates adaptive responses to abiotic and biotic stresses
(Chernys and Zeevaart, 2000; Shang etal., 2010). At present,
major progress has been made in research into the role of
ABA in the regulation of fleshy fruit ripening (Rodrigo etal.,
2006; Zhang et al., 2009a, b; Giribaldi et al., 2010). These
physiological processes controlled by ABA are primarily
regulated by the bioactive ABA pool size, which is thought to be
maintained not only by its biosynthesis, but also by its
catabolism (Sawada etal., 2008). The ABA metabolic pathway has
been established by genetic approaches. ABA is synthesized
de novo from a C40 carotenoid. The carotenoid-biosynthetic
pathway begins with the formation of phytoene from two
molecules of geranylgeranyl diphosphate (GGPP) in the
central isoprenoid pathway. Four desaturation steps give rise to
lycopene; cyclizations at both ends of the lycopene molecule
produce -or -carotene, which undergo hydroxylation at
C3 and C3 to form the xanthophylls lutein and zeaxanthin,
The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
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respectively. An important phase of ABA biosynthesis is
initiated in plastids with the hydroxylation and epoxidation of the
-carotene to produce the all-trans xanthophylls zeaxanthin
and violaxanthin. Violaxanthin is then converted into
9-cisepoxyxanthophylls, which are oxidatively cleaved by
9-cisepoxycarotenoid dioxygenase (NCED) to yield xanthoxin,
the first C15 intermediate of ABA biosynthesis. Xanthoxin
exits the plastid into the cytosol where it is oxidized in two
further steps to form ABA (Schwartz et al., 2003; Taylor
etal., 2005). In addition, the ABA metabolic pathway plays
an important role in the regulation of ABA levels (Cutler and
Krochko, 1999; Schwartz et al., 2003). The hydroxylation
pathway is the main ABA catabolic pathway. In the
hydroxylation pathway, among three different methyl groups, C8
is the predominant position for the hydroxylation reaction,
which is mediated in Arabidopsis by proteins encoded by the
CYP707A gene family (Saito etal., 2004; Kushiro etal., 2004).
Dihydroxyphaseic acid (DPA) may be the major metabolite
of ABA (Setha etal., 2005). The 8-hydroxylation is reported
to be the major regulatory step in many physiological events
controlled by ABA (Kushiro etal., 2004; Saito etal., 2004).
Recently, a major breakthrough in the field of ABA signalling
was achieved with the identification of the PYR/PYL/RCAR
protein family, the type 2C protein phosphatases (PP2Cs),
and subfamily 2 of the SNF1-related kinases (SnRK2s) (Ma
etal., 2009; Park etal., 2009; Melcher etal., 2009; Santiago
etal., 2009). To elucidate the mechanism of ABA action, it
is necessary to identify all the components involved in ABA
homeostasis, including the functional components in ABA
metabolic pathways, signal transduction, and transport.
In tomato (Solanum lycopersicum), three NCED genes have
been isolated, and expression analysis indicated that, among
them, SlNCED1 may regulate ABA biosynthesis in fruit (Sun
etal., 2012a, b). However, the molecular evidence remains to
be elucidated. To address this, a key step in ABA biosynthesis,
NCED was targeted for inhibition via RNAi in tomato fruit
two years previously. Four independent transgenic plants
were evaluated (Sun et al., 2012a, b). Our data showed that
ABA potentially regulated the development and ripening of
tomato fruit. In addition, ABA could control, at least in part,
the production and effects of ethylene in climacteric tomato
fruit. In recent years, tobacco rattle virus-induced gene
silencing (VIGS) has been used as a rapid gene function assay
system in molecular biological studies of fleshy fruit (Fu etal.,
2006; Hoffmann et al., 2006; Li et al., 2013). In this study,
we further identify the function of three NCED and three
CYP707A genes by VIGS. Results show that SlNCED1 and
SlCYP707A2 are key genes in the regulation of ABA level
during development and ripening of tomato fruit.
Materials and methods
Construction of the viral vector and agroinoculation
The pTRV1 and pTRV2 virus-induced gene silencing vectors
(described by Liu et al., 2002) were kindly provided by YL Liu
(School of Life Science, Tsinghua University, Beijing, China).
A 456 bp cDNA fragment of an NCED or CYP707A gene was
amplified using primers (Table 1). The amplified fragment was
cloned into EcoRI/SacI-digested pTRV2. Agrobacterium
tumefaciens strain GV3101 containing pTRV1, pTRV2, and the
pTRV2derivative pTRV2-NCED/CYP707A were used for RNAi. Thirty
fruits from ten independent plants grown in the greenhouse were
selected for inoculation, and each basal pedicel or one side of fruit
was injected with the NCED/CYP707A-RNAi TRV vector at the
maturation green stage. The fruits were evaluated 312 days after
treatment.
Plant materials
Tomatoes (Solanum lycopersicum L.cv. JiaBao) were grown under
standard greenhouse conditions (25 5C and 70% humidity under
a 14 h/10 h light/dark regime). Fruit ripening stages were divided
according to the days after flowering (DAF) and fruit colour:
immature green (IM), 15 DAF; mature green (MG), 30 DAF; breaker 0
(B0), 34 DAF; breaker 1 (B1), 35 DAF; breaker 2 (B2), 36 DAF;
turning 0 (T0), 37 DAF; turning 3 (T3), 40 DAF; red ripe (R), 42
DAF; and over ripe (OR), 45 DAF. Ten fruits were harvested at each
stage and immediately frozen in liquid nitrogen. They were then
powdered, mixed, and stored at 80oC until further use. Tom
tomatoes were also grown in the same greenhouse.
Dehydration treatment offruits
In order to evaluate the effect of dehydration stress, 60 fruits were
harvested at the MG stage and divided evenly into two groups. The
first group (control) was stored at 20C under high relative humidity
(RH) (95%) which included 10 control fruits, 10
SlNCED1-RNAitreated fruits, and 10 SlCYP707A2-RNAi-treated fruits. The second
group (dehydration stress) was stored at the same temperature and
subjected to the same treatments, but under low RH (55%,
dehydrated fruits). The ABA content and expression of related genes in
the pulp were determined 0, 3, 5, and 7 d after the treatment of
fruits and 0, 1, and 2 d after the treatment of sepals. Every single
fruit was weighed immediately after harvest and then weighed again
before sampling for calculation of water loss rate. Water loss rate
is calculated as the ratio of the decreased fruit weight to the initial
fruit weight.
ABA or NDGA treatment
Sixty fruits harvested at the MG stage were divided into two groups
(n=30 for each group), and immediately soaked in 100M ABA
(Sigma, A1049, USA) (group I) or distilled water (group II,
control) for 10 min under low vacuum. The fruits were then placed in
a tissue culture room at 25C and 95% RH. After 0, 1, and 3d, the
fruits were sampled, frozen with liquid nitrogen, powdered, mixed,
and stored at 80oC for further use. Different treatments with
Table1. Specific primers used for VIGS in this study
Oligonucleotides (5-3)
CGGGAATTCTAGTTACGCTTGCCGTTTCACTGAA
CGGGAGCTCTCAGGAATGACGACGAAGTTCTCAG
CGGGAATTCACAAGACGACAACTACTTTCACCCT
CGGGAGCTCTCTAGAGTCATGGCATTTACAATTTG
CGGGAATTCTCCACGACCCGAATAAAGTATCT
CGGGAGCTCGTCTTGTTTACTTGTCCCGCTTC
CGGGAATTCCATTTGGATGGTCATGTTAAGGA
CGGGAGCTCCAAATAACTTTCTGTTCAGCCTTGA
CGGGAATTCTCTTTGCAGCTCGAGACACTACTGC
CGGGAGCTCTCCAGCTTGGCTAAGTCATTCCCT
CGGGAATTCTTTCAAGACTCACATATTGGGATG
CGGGAGCTCTAGCACCTCTTTAGATCCTCCCT
nordihydroguaiaretic acid (NDGA) were the same as with ABA;
NDGA concentration was 200M.
Quantitative real-time PCR analysis
Total RNA was isolated from tomato samples using the hot borate
method (Wan and Wilkins, 1994). Genomic DNA was eliminated
using an RNase-free DNase Ikit (Takara, China) according to the
manufacturers recommendations. For every RNA sample, quality
and quantity were assessed by agarose gel electrophoresis. cDNA
was synthesized from total RNA using the PrimeScriptTM RT
reagent kit (Takara) according to the manufacturers recommendations.
Primers used for real-time PCR are listed in Supplementary Table S1
and were designed using Primer 5 software
(http://www.premierbiosoft.com/). SAND was used as an internal control gene, and the
stability of its expression was tested in preliminary studies (Sun etal.,
2011). All primer pairs were tested by PCR. The presence of a single
product of the correct size for each gene was confirmed by agarose
gel electrophoresis and double-strand sequencing (Invitrogen). The
amplified fragment of each gene was subcloned into the pMD18
T vector (Takara), and used to generate standard curves through
serial dilution. The real-time PCR was performed using a
RotorGene 3000 system (Corbett Research, China) with SYBR Premix
Ex TaqTM (Takara). Each 20l reaction solution contained 0.8l of
primer mixer (containing 4M of each forward and reverse primer),
1.5 l cDNA template, 10 l SYBR Premix Ex TaqTM (2X) mixer,
and 7.7l water. Reactions were performed under the following
conditions: 95C for 30 s (one cycle), 95C for 15 s, 60C for 20 s, and
72C for 15 s (40 cycles). The changes of relative fold expression
were calculated using the relative two standard curves method with
Rotor-Gene 6.1.81 software (Invitrogen).
Determination of ABA content
For ABA extraction, 1.0 g of pulp was ground in a mortar and
homogenized in the extraction solution (80% v/v methanol). Extracts
were centrifuged at 10 000g for 20 min. The supernatant was eluted
through a Sep-Pak C18 cartridge (Waters, www.waters.com) to
remove polar compounds, and then were stored at 20C for ELISA.
The stepwise procedure for indirect ELISA of ABA was as follows:
each well of a microtitre plate was pre-coated with ABABSA
conjugater diluted in coating buffer according to the instructions of
the manufacturer (ELISA kit for ABA, College of Agronomy and
Biotechnology, China Agricultural University). Then, to each well,
was added 50 l standard or sample in assay buffer (8.0 g NaCl,
0.2 g KH2PO4, 2.96 g Na2HPO412 H2O, 1.0 ml Tween 20, and 1.0 g
gelatin, added to 1.000 ml water), followed by 50l ABA antibody
(Invitrogen) diluted 1:2000 in assay buffer. The plates were
incubated for 0.5 h at 37C and then washed four times with scrubbing
buffer (which contained the same ingredients as assay buffer, but
without gelatin). Anti- mouse IgG coupled to alkaline phosphatase
(100 ml of a 1:1000 dilution) was added to each well, and the plates
were incubated for 0.5 h at 37C. The plates were washed as above,
and then 100l of a 12 mg ml1 o-phenylenediamine substrate
solution and 0.04% by volume of 30% v/v hydrogen peroxide in substrate
buffer (5.10 g C6H8O7H2O, 18.43 g Na2HPO412 H2O, and 1.0 ml
Tween 20, added to 1000 ml water) were added to each well. After
1015 min, 50l of 2.0 mol l1 H2SO4 was added to each well to
terminate the reaction. The absorbance was measured at 490 nm using
a Thermo Electron (Labsystems) Multiskan MK3 (Pioneer, www.
pioneerbiomed.com). The concentration of ABA in the sample was
calculated from log B/B0-transformed standard curve data, where
B and B0 are the absorbance values with or without the competing
antigen, respectively.
Determination of ethylene production
The ethylene production of the fruit was measured by enclosing
three fruits in 1.0 l airtight containers for 2 h at 20C, withdrawing
1 ml of the headspace gas, and injecting it into a gas chromatograph
(Agilent model 6890N) fitted with a flame ionization detector and an
activated alumina column. Fresh tissues from each fruit were frozen
in liquid nitrogen and stored at 80C until use.
Determination of fruit firmness
Fruits were harvested from all of the plants in each of three
replicate plantings at the different ripening stages. Flesh firmness was
measured after the removal of fruit skin on three sides of each fruit
using a KM-model fruit hardness tester (Fujihara). The strength of
flesh firmness was recorded in kg cm1. Compression of each fruit
was measured three times, and the average of the maximum force
was used.
Expression patterns of ABA metabolic genes in pulp
during fruit development and in response to application
of exogenous ABA and dehydrationstress
Within the SlNCED gene family, the expression of SlNCED1
was the highest in pulp. SlNCED1 decreases from 10 days
after full bloom (DAFB) to the MG stage, then it increased
sharply and peaked at the turning stage; after that it declined
to a low level at the OR stage (Fig.1A). The expression
variation of SlNCED2 was generally decreased from 10 DAFB
to fruit ripening. The expression of SlNCED3 was very
low through fruit development and ripening. Among the
SlCYP707A gene family, the expression of SlCYP707A2
(Fig. 1B) exhibited a fluctuant expression pattern with four
peaks during development, and then it increased rapidly
during ripening. Compared to SlCYP707A2, the expression of
SlCYP707A1 and SlCYP707A3 (Fig.1B) was very low
during fruit development and ripening. In addition, expression
of ABA metabolic genes in response to exogenous ABA
treatment and dehydration in tomato fruits was tested. For
SlNCED1, expression was significantly increased by
exogenous ABA and dehydration at 2days after treatment (DAT)
(Fig. 1C, E). The expression of SlNCED2 and SlNCED3
was also significantly increased in the water stressed and
ABA-treated tomato fruits (Fig. 1C, E). For SlCYP707A2,
expression was downregulated in the fruits under both ABA
treatment and dehydration at 1 DAT (Fig.1D, F), and then it
significantly increased at 2 DAT (Fig.1D, F). The expression
of SlCYP707A1 and SlCYP707A3 was significantly
downregulated under both water stress and ABA treatment at 1
DAT, but upregulated at 2DAT.
Silencing of the SlNCED1 gene suppresses tomato
fruit ripening
To examine the role of SlNCEDs, the tobacco rattle virus
(TRV) vector was used to suppress the expression of SlNCEDs
(Fig.2). When the SlNCED1-RNAi TRV vector was injected
into the basal pedicel of 15 fruits (cv. Tom) attached to the
plant at the MG stage, fruit ripening slowed down, and the
entire fruit turned orange (Fig. 3D, E,F) instead of red as
in the control (Fig.3A). Ten days after the SlNCED1-RNAi
TRV vector was injected, the fruits did not show normal
ripening (Fig.3D, E, F) as in the control (Fig.3A). Compared
to SlNCED1-RNAi-treated fruits, there were no significant
differences in colouring between SlNCED2/3-RNAi-treated
fruits and control fruits (Fig.3B, C) during fruit ripening and
in response to dehydration stress. The SlNCED1-RNAi TRV
vector was also injected into the attached fruits (cv. JiaBao)
at the MG stage. 12 d after injection, control fruits turned red
(Fig. 3M, N); however, for SlNCED1-RNAi-treated fruits,
parts of the peel and placenta inside the fruit did not turn
red as in the control (Fig.3K, L). The water loss of the sepal
in the SlNCED1-RNAi-treated fruits was quicker than in the
control under the same conditions. Meanwhile, the wilting of
the sepals was more serious than that of the control under the
same conditions.
SlNCED1-RNAi treatment alters the expression of
genes involved in ABA-responsivegenes
In SlNCED1-RNAi-treated fruits, expression of SlNCED1
was markedly downregulated to 19% of the control while
expression of SlNCED2/SlNCED3 was downregulated/
upregulated (Fig. 4A, B, C). In fruits with the same
treatment, the expression of SlCYP707A1/2 was
downregulated, while the expression of SlCYP707A3 was upregulated
(Fig.4D, E, F). Among the ABA-signalling genes,
including those of the PYR/PYL/RCAR protein family (SlPYL1),
type 2C protein phosphatases (SlPP2C1), and subfamily 2
of SNF1-related kinases (SlSnRK2.2) (Ma etal., 2009; Park
et al., 2009), SlPYL1and SlSnRK2.2 were downregulated
while SlPP2C1 was upregulated (Fig.4G, H, I). As shown
in Fig.5A, in SlNCED1-RNAi-treated fruits, the ABA
content was 21% of the control at the turning stage (7 d after
MG); moreover, fruits couldnt become fully red and this
incomplete colouring couldnt be rescued by the application
of exogenous ABA (although ABA contents were increased
by application of exogenous ABA) (Fig.5A). Expression of
SlPYL1 and SlSnRK2.2 in SlNCED1-RNAi-treated fruits
couldnt be increased by application of exogenous ABA
(Fig.5B).
Fig.4. Expression of ABA-responsive genes in both control and SlNCED1-RNAi-treated fruit during development and ripening of tomato. The JiaBao
fruits were injected with SlNCED1-RNAi TRV vectors at THE MG stage. Fruits were sampled 0 d (MG), 5 d (B), 7 d (T), 9 d (HR), and 12days (OR) after
the inoculation, respectively. SAND mRNA was used as the internal control. Three biological replicates (n=3) were used for each analysis. *P value t-test
< 0.05; **P value t-test < 0.001. Error bars are SE.
SlNCED1-RNAi treatment alters the expression of
genes involved in ripening-relatedgenes
Several ripening-related physiological parameters were
measured, including fruit firmness, solid soluble content,
and lycopene content. As shown in Fig. 6, in
SlNCED1RNAi fruits, the trends were for a decrease in solid soluble
content and lycopene content, but an increase in fruit
firmness. To clarify the role of SlNCED1 in the regulation of
tomato fruit ripening, several ripening-related genes were
examined in both SlNCED1-RNAi-treated fruits and
control fruits. SlBcyc, SlPSY1 and SlPDS encode lycopene
-cyclase, phytoene synthetase, and phytoene
dehydrogenase, respectively. Relative quantitative real-time PCR
analysis showed that the expression of all these genes was
downregulated (Fig. 7H, I) except for SlBcyc, which was
upregulated (Fig.7J). In addition, we examined the
expression of genes encoding cell-wall hydrolases. In
SlNCED1RNAi fruit, genes encoding polygalacturonase (SlPG1),
expansin (SlEXP1), and xyloglucan endotransglycosylase
(SlXET16) were all significantly downregulated during fruit
ripening compared to the control (Fig.7E, F, G). Relative
expression analysis showed that the expressions of SlACS2
[encoding 1-aminocyclopropane-1-carboxylic acid (ACC)
synthase], SlACO1 (encoding ACC oxidase), and SlETR3
(involved in the ethylene response), were upregulated in
the SlNCED1-RNAi fruits (Fig.7A, B, C). The expression
of SlERF2 was significantly downregulated in
SlNCED1RNAi fruits (Fig.7D). Ethylene release was upregulated at
turning stage, but downregulated at harvest red stage
compared to the control (Fig.7K).
Silencing of the SlCYP707A2 gene promotes tomato
fruit colouring
To clarify the role of SlCYP707A2 in the regulation of ABA
levels during fruit ripening, ABA levels and ABA-responsive
genes were examined in both RNAi-treated and control fruits.
When the SlCYP707A2 -RNAi TRV vector was injected into
the 15 fruits attached to the plant at the MG stage, the fruits
could become red quicker (Fig. 8F), and ripening was also
faster than in the control (Fig. 8G). In
SlCYP707A1/2/3RNAi-treated Tom fruits (Fig.8B, C, D), fruit ripening
(Fig.8B, C, D) was the same as with control fruit (Fig.8A) at
harvest stage. In SlCYP707A2-RNAi-treated fruits, the ABA
content was higher than the control at 5 and 7 d after MG,
and fruit ripening couldnt be inhibited by the application of
NDGA, which delayed the control fruit ripening (Fig.9J).
SlCYP707A2-RNAi treatment alters the expression of
genes involved in ABA-responsivegenes
In the SlCYP707A2-RNAi-treated fruits, expression
of SlCYP707A1 was downregulated, but SlCYP707A3
Fig.5. Changes in ABA content, SlPYL1 and SlSnRK2.2 expression,
and numbers of fully red fruit 9 d after exogenous ABA treatment in both
control and SlNCED1-RNAi-treated fruit. 78 JiaBao fruits were equally
divided into two groups at the MG stage and then injected with 1 ml ABA
(100 mM) for group 1, or distilled water for group 2 (control). 30 fruits
were used for the investigation of number of fully red fruits and others
were sampled at 0, 5, 7, and 9 d after ABA treatment, respectively, for the
determination of ABA content and gene expression. SAND mRNA was
used as the internal control. Three biological replicates (n=3) were used
for each analysis. *P value t-test < 0.05; **P value t-test < 0.001. Error
bars are SE.
was upregulated; however, SlCYP707A2 was markedly
downregulated to 18% of the control (Fig.9D, E, F).
The expression of SlNCED1/3 was upregulated, while
SlNCED2 was downregulated in SlCYP707A2-RNAi
fruits (Fig.9A, B, C). The expression of SlPYL1 and
SlSnRK2.2 in ABA signalling was upregulated, while
SlPP2C1 expression was downregulated in
SlCYP707A2RNAi fruit (Fig.9G, H, I).
SlCYP707A2-RNAi treatment alters the expression of
genes involved in ripening-relatedgenes
As shown in Fig.6, fruit firmness was lower than that of the
control in SlCYP707A2-RNAi fruits, while the soluble solid
content and lycopene content did not show significant
differences compared to the control fruits. In SlCYP707A2-RNAi
fruits, genes encoding polygalacturonase (SlPG), expansin
(SlEXP) and xyloglucan endotransglycosylase (SlXET16)
Fig.6. Changes in fruit firmness, solid soluble, and lycopene content
in control, SlNCED1-RNAi-treated, and SlCYP707A2-RNAi-treated
fruits. Every nine JiaBao fruits from control, SlNCED1-RNAi-treated,
and SlCYP707A2-RNAi-treated fruits, respectively, were harvested at
the harvest red stage. Three biological replicates (n=3) were used for
each analysis. *P value t-test < 0.05; **P value t-test < 0.001. Error
bars are SE.
were upregulated at breaker and turning stages; however,
there were no significant changes compared to the control
fruits at the harvest stage (Fig.10E, F, G). In the lycopene
synthesis pathway, compared with the control, the relative
expression levels of SlPSY1 and SlPDS were higher, but
SlBcyc was lower, at breaker and turning stages (Fig.10H,
I, J). With respect to ethylene, relative expression analysis
showed that the expression of SlACS2 [encoding (ACC)
synthase], SlACO1 (encoding ACC oxidase), and SlETR3
(involved in the ethylene response), which were consistent
with ethylene release (Fig.10K) in the SlCYP707A2-RNAi
fruits, were upregulated at breaker and turning stages;
however, there was no significant difference at harvest
stage compared to control fruits (Fig. 10J). The expression
of the SlERF2 was upregulated at the T and HR stages.
SlCYP707A2-RNAi-treated fruits during the harvest stage
were unusual, with uneven colouring in pulp compared to
control fruits.
Dehydration of SlNCED1/
SlCYP707A2-RNAi-treatedfruits
Both control and SlNCED1/SlCYP707A2-RNAi-treated
fruits were harvested 6 d after the
SlNCED1/SlCYP707A2RNAi treatments (Fig. 11). The fruits were then incubated
in the laboratory (20C, 50% relative humidity). As shown
in Fig.11, the weight loss rate in SlNCED1-RNAi fruits was
higher than the control fruit 36 d after dehydration. However,
there was not a large difference in the rate of water loss
comparing control and SlCYP707A2-RNAi fruit (Fig.11A). The
water loss of the sepal in the SlNCED1/SlCYP707A2-RNAi
treated fruit was also similar to that of fruit under the same
conditions (Fig.11B).
Discussion
NCED and CYP707A are generally encoded by a small
gene families, respectively (Krochko etal., 1998; Burbidge
etal., 1999; Zhang etal., 2009a). Among the three NCED
genes in tomato, SlNCED1 may play a primary role in
regulating ABA biosynthesis during fruit ripening (Fig. 1A)
in response to ABA application (Fig.1E) and dehydration
(Fig.1C). Besides biosynthesis, catabolism of ABA is also
an important way of regulating ABA levels (Kushiro etal.,
2004; Li et al., 2011; Ren et al., 2011). A similar result to
the reports of Ren et al. (2010) and Wang et al. (2013)
was obtained in this work: the expression of SlCYP707A2
was higher than SlCYP707A1 and SlCYP707A3, and was
opposite to the change of ABA content during
development (Fig.1B) in response to ABA treatment (Fig.1F) and
dehydration (Fig. 1D). Previously, to suppress SlNCED1
specifically in tomato fruits, we used an RNA interference
bars are SE.
Fig.11. Effects of dehydration stress on control, SlNCED1-RNAi-treated,
and SlCYP707A2-RNAi-treated fruits. Fruits at the MG stage were placed into
an incubator: control, 25oC and 90% relative humidity; dehydration, 25oC and
45% relative humidity. Fruits were sampled 0, 3, and 6 d after dehydration
treatment (DADT), respectively. The sepal samples were collected at 1 and
2 DADT. Three biological replicates (n=3) were used for each analysis. *P value t-test < 0.05; **P value t-test < 0.001. Error bars are SE.
Supplementary material
Supplementary data can be found at JXB online.
Supplementary Table S1. Specific primer sequences used
for real-time quantitative PCR.
This work was partly supported by the 973 Programme
2012CB113900 to Yang-Dong Guo.